Cigarette smoking causes approximately 30% of all cancer death in the U.S. It is responsible for 85% of the 160,000 lung cancer deaths expected in 1998. Snuff-dipping is an acknowledged cause of oral cavity cancer and the use of moist snuff in the U.S. has increased remarkably in recent years. In spite of advances in tobacco control and smoking cessation, there are still 48 million smokers in the U.S., about 25% of the adult population. Approximately 500 billion cigarettes and 121 million pounds of smokeless tobacco were consumed in 1997 in the U.S. Tobacco-specific N-nitrosamines are formed from tobacco alkaloids during the curing and processing of tobacco products. Two of these, 4- (methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) and N- nitrosonornicotine (NNN), are present in substantial quantities and are strong carcinogens. NNK is a potent pulmonary carcinogen in rodents and also induces tumors of the pancreas, nasal mucosa, and liver. NNN causes tumors of the esophagus and nasal mucosa in rats and respiratory tract tumors in mice and hamsters. A mixture of NNK and NNN induces oral tumors in rats. Based on their carcinogenic activities, the extensive data on the occurrence of NNK and NNN in tobacco products, and on biochemical studies, it is our hypothesis that these compounds play a significant role as causative factors in human cancers associated with tobacco use. Although there are parallels between NNK and NNN metabolism in rodents and humans which support our hypothesis, there are key aspects which must be explored to further evaluate our hypothesis. Therefore, our specific aims are: 1) investigate stereoselectivity on the metabolism of NNK and NNN by delineating differences in the formation and further metabolism of enantiomers of the major NNK metabolite NNAL, determining differences in the stereoselectivity of metabolic activation of NNK, and examining differences in the metabolic activation and carcinogenicity of NNN enantiomers; 2) investigate DNA and protein pyridyloxo-butylation by NNK and NNN by elucidating the structures of the major adducts formed with DNA and hemoglobin; and 3) investigate the metabolism of NNK and NNN in humans by determining levels of NNN metabolites in human urine, characterizing previously unknown NNN metabolites, and examining NNK metabolites in the urine of snuff-dippers to determine their persistence after cessation and their amounts as a function of dose. The overall theme of this proposal is mechanistic studies of NNK and NNN metabolism and adduct formation, in order to further evaluate the carcinogenic potential of NNK and NNN in humans exposed to tobacco products. We believe that an understanding of the mechanisms by which tobacco smoke carcinogens cause cancer will lead to new insights on individual cancer susceptibility and development of innovative strategies to prevent tobacco related cancers.